In the last report, we characterized a parity-induced mammary epithelial cell population, which possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of beta-galactosidase (LacZ) as a result of mammary-specific activation of a reporter gene through cre-lox recombination during pregnancy. We used this experimental model to determine if testicular cells would alter their cell fate upon interaction with the mammary gland microenvironment during pregnancy, lactation and involution. Adult testicular cells,neural stem cells (both fetal and adult), erbB2-induced mouse mammary tumor cells and human embryonal cancer cells, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. This approach allowed for the growth of mammary tissue from the injected cells.The tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we demonstrated that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. FISH confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric DNA, as well as immunochemistry for human specific breast epithelial cytokeratins and human specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66-660 fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results demonstrate that human embryonal carcinoma-derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When MMTV-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed mammary epithelial cells during regenerative growth. In the presence of non-transformed MECs (50:1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells demonstrate the capacity to differentiate into normal mammary cell types including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth. Amphiregulin (AREG) is a ligand for the epidermal growth factor receptor (EGFR) and in studies of estrogen receptor alpha null (ERalpha -/-) mice was shown to mediate paracrine signaling by estradiol through ERalpha+ mammary epithelial cells. AREG function is required for ductal morphogenesis and mediates estrogen actions in vivo. Thus, AREG has emerged as an essential growth factor during mammary gland growth and differentiation The COMMA-D beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary cells including the stem cell capacity to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile host. These cells display additional stem cell properties including nuclear label retention and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDbetageo cells, structures that represent expansion of progenitor cells. Our evidence indicates that the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG-/- mammary epithelial cells. These data demonstrate AREG is essential for the function of mammary progenitor cells in vitro. The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When MMTV-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed mammary epithelial cells during regenerative growth. In the presence of non-transformed MECs (50:1),, tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells demonstrate the capacity to differentiate into normal mammary cell types including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.